Late graft failure of pig-to-rhesus renal xenografts has features of glomerulopathy and recipients have anti-swine leukocyte antigen class I and class II antibodies.

Prolonged survival in preclinical renal xenotransplantation demonstrates that early antibody mediated rejection (AMR) can be overcome. It is now critical to evaluate and understand the pathobiology of late graft failure and devise new means to improve post xenograft outcomes. In renal allotransplantation the most common cause of late renal graft failure is transplant glomerulopathy-largely due to anti-donor MHC antibodies, particularly anti-HLA DQ antibodies. We evaluated the pig renal xenograft pathology of four long-surviving (>300 days) rhesus monkeys. We also evaluated the terminal serum for the presence of anti-SLA class I and specifically anti-SLA DQ antibodies. All four recipients had transplant glomerulopathy and expressed anti-SLA DQ antibodies. In one recipient tested for anti-SLA I antibodies, the recipient had antibodies specifically reacting with two of three SLA I alleles tested. These results suggest that similar to allotransplantation, anti-MHC antibodies, particularly anti-SLA DQ, may be a barrier to improved long-term xenograft outcomes.

Xenoreactive antibodies in α-granules of human platelets bind pig liver endothelial cells.

Pig liver xenotransplantation is limited by a thrombocytopenic coagulopathy that occurs immediately following graft reperfusion. In vitro and ex vivo studies from our lab suggested that the thrombocytopenia may be the result of a species incompatibility in platelet glycosylation. Realization that platelet α-granules contain antibodies caused us to reevaluate whether the thrombocytopenia in liver xenotransplantation could occur because IgM and IgG from inside platelet α-granules bound to pig liver sinusoidal endothelial cells (LSECs). Our in vitro analysis of IgM and IgG from inside α-granules showed that platelets do carry xenoreactive antibodies that can bind to known xenoantigens. This study suggests that thrombocytopenia occurring following liver xenotransplantation could occur because of xenoreactive antibodies tethering human platelets to the pig LSEC enabling the platelet to be phagocytosed. These results suggest genetic engineering strategies aimed at reducing xenoantigens on the surface of pig LSEC will be effective in eliminating the thrombocytopenia that limits survival in liver xenotransplantation.

Patients on the Transplant Waiting List Have Anti-Swine Leukocyte Antigen Class I Antibodies.

Organ supply remains inadequate to meet the needs of many patients who could benefit from allotransplantation. Xenotransplantation, the use of animals as organ donors, provides an opportunity to alleviate this challenge. Pigs are widely accepted as the ideal organ donor, but humans and nonhuman primates have strong humoral immune responses to porcine tissue. Although carbohydrate xenoantigens have been studied intensively, the primate Ab response also targets class I and class II swine leukocyte Ags (SLAs). Human Abs that recognize HLAs can cross-react with SLA molecules because epitopes can be shared across species. However, ∼15% of people may also exhibit Abs toward class II SLAs despite lacking Abs that also recognize class II HLAs. Here, we extend these studies to better understand human Ab responses toward class I SLAs. When tested against a panel of 18 unique class I SLA proteins, 14 of 52 sera samples collected from patients in need of an organ transplant contained Abs that bound class I SLAs. Class I SLA-reactive sera may contain IgM only, IgG, only, or IgM and IgG capable of recognizing the pig proteins. The presence of class I HLA-reactive Abs was not essential to generating anti-class I SLA Ig. Last, anti-class I SLA reactivity varied by serum; some recognized a single SLA allele, whereas others recognized multiple class I SLA proteins.

Thermal oscillation in the hybrid Si3N4 - TiO2 microring.

The hybrid microcavity composed of different materials shows unique thermal-optical properties such as resonance frequency shift and small thermal noise fluctuations with the temperature variation. Here, we have fabricated the hybrid Si3N4 - TiO2 microring, which decreases the effective thermo-optical coefficients (TOC) from 23.2pm/K to 11.05pm/K due to the opposite TOC of these two materials. In this hybrid microring, we experimentally study the thermal dynamic with different input powers and scanning speeds. The distorted transmission and thermal oscillation are observed, which results from the non-uniform scanning speed and the different thermal relaxation times of the Si3N4 and the TiO2. We calibrate the distorted transmission spectrum for the resonance measurement at the reverse scanning direction and explain the thermal oscillation with a thermal-optical coupled model. Finally, we analyse the thermal oscillation condition and give the diagram about the oscillation region, which has significant guidance for the occurrence and avoidance of the thermal oscillation in practical applications.

[Developing biomass estimation models of young trees in typical plantation on the Qinghai-Tibet Plateau, China.] / é’è—é«˜åŽŸå ¸åž‹äººå·¥æž—å¹¼æ ‘ç”Ÿç‰©é‡æ¨¡åž‹æž„å»º.

Calculation of forest biomass is the basis for global carbon stock estimation, which has been included in national forest inventory projects. The volume-derived biomass method is generally used for trees with diameter at breast height (DBH) larger than 5 cm in most forest carbon sink measurement, which omits young trees (diameter at breast height <6 cm, height >0.3 m) and thus may underestimate ecosystem carbon sink capacity. Based on the biomass data of 137 young trees in five typical plantations on the Tibetan Plateau, independent biomass models were developed using the weighted generalized least squares method, with basic diameter as the predictor instead of DBH. Additive biomass models of controlling directly by proportion functions and controlling by the sum of equations were selected. Additive biomass models for the whole plant and each component were developed by applying weighted nonlinear seemingly uncorrelated regression. The results showed that the binary additive biomass model (R2 reached 0.90-0.99) performed better than the monadic biomass models and independent biomass models for the estimation of total biomass. For different tree species, two forms of the additive models had their own advantages, with neglectable difference in accuracy. From the perspective of forestry production, models of controlling directly by proportion functions were more practical. From the perspective of predictors extraction by remote sensing technology, suitable young tree biomass models were developed for remote sensing estimation. In this study, the additive model had high overall fitting accuracy and could accurately estimate the whole plant and component biomass of young trees in similar climatic environments.

Numerical characterization of soliton microcomb in an athermal hybrid Si3N4-TiO2 microring.

We theoretically investigate the athermal constructions to cancel the thermorefractive effect of a hybrid Si3N4-TiO2 microring, which merges two materials with opposite thermo-optical coefficients (TOCs). The analytical and numerical results predict that the thermorefractive effect can be reduced under the appropriate parameters. In addition, the soliton state is easily accessed under the athermal condition. The thermorefractive noise due to the fluctuation of the microresonator temperature caused by the heat exchange between the microresonator and the surrounding environment is also suppressed by one order of magnitude, which is critical for the potential applications of soliton microcombs, such as spectroscopy, optical clocks and microwave generation.

In vivo fluorescent screening for HPPD-targeted herbicide discovery.

BACKGROUND: 4-Hydroxyphenylpyruvate dioxygenase (HPPD), playing a critical role in vitamin E and plastoquinone biosynthesis in plants, has been recognized as one of the most important targets for herbicide discovery for over 30 years. Structure-based rational design of HPPD inhibitors has received more and more research interest. However, a critical challenge in the discovery of new HPPD inhibitors is the common inconsistency between molecular-level HPPD-based bioevaluation and the weed control efficiency in fields, due to the unpredictable biological processes of absorption, distribution, metabolism, and excretion. RESULTS: In this study, we developed a fluorescent-sensing platform of efficient in vivo screening for HPPD-targeted herbicide discovery. The refined sensor has good capability of in situ real-time fluorescence imaging of HPPD in living cells and zebrafish. More importantly, it enabled the direct visible monitoring of HPPD inhibition in plants in a real-time manner. CONCLUSION: We developed a highly efficient in vivo fluorescent screening method for HPPD-targeted herbicide discovery. This discovery not only offers a promising tool to advance HPPD-targeted herbicide discovery, but it also demonstrates a general path to develop the highly efficient, target-based, in vivo screening for pesticide discovery. © 2022 Society of Chemical Industry.

Deferoxamine/magnesium modified ß-tricalcium phosphate promotes the bone regeneration in osteoporotic rats.

Recently, Deferoxamine (DFO) and magnesium (Mg) have been identified as critical factors for angiogenesis and bone formation. However, in current research studies, there is a lack of focus on whether DFO plus Mg can affect the regeneration of ß-tricalcium phosphate (ß-TCP) in osteoporosis and through what biological mechanisms. Therefore, the present work was aimed to preparation and evaluate the effect of Deferoxamine/magnesium modified ß-tricalcium phosphate promotes (DFO/Mg-TCP) in ovariectomized rats model and preliminary exploration of possible mechanisms. The MC3T3-E1 cells were co-cultured with the exudate of DFO/Mg-TCP and induced to osteogenesis, and the cell viability, osteogenic activity were observed by Cell Counting Kit-8(CCK-8), Alkaline Phosphatase (ALP) staining, Alizarin Red Staining (RES) and Western Blot. In vitro experiments, CCK-8, ALP and ARS staining results show that the mineralization and osteogenic activity of MC3T3-E1increased significantly after intervention by DFO/Mg-TCP, as well as a higher levels of protein expressions including VEGF, OC, Runx-2 and HIF-1α. In vivo experiment, Micro-CT and Histological analysis evaluation show that DFO/Mg-TCP treatment presented the stronger effect on bone regeneration, bone mineralization and biomaterial degradation, when compared with OVX+Mg-TCP group and OVX+TCP group, as well as a higher VEGF, OC, Runx-2 and HIF-1α gene expression. The present study indicates that treatment with DFO/Mg-TCP was associated with increased regeneration by enhancing the function of osteoblasts in an OVX rat.

Thermal tuning of mode crossing and the perfect soliton crystal in a Si3N4 microresonator.

Dissipative Kerr solitons in high quality microresonators have attracted much attention in the past few years. They provide ideal platforms for a number of applications. Here, we fabricate the Si3N4 microring resonator with anomalous dispersion for the generation of single soliton and soliton crystal. Based on the strong thermal effect in the high-Q microresonator, the location and strength of the avoided mode crossing in the device can be changed by the intracavity power. Because the existence of the avoided mode crossing can induce the perfect soliton crystal with specific soliton number, we could choose the appropriate pumped resonance mode and appropriate pump power to obtain the perfect soliton crystals on demand.

A new method of monitoring catheter-directed thrombolysis for deep venous thrombosis-application of D-dimer and fibrinogen testing.

BACKGROUND: Catheter-directed thrombolysis (CDT) is one of the main treatment methods for acute deep venous thrombosis (DVT), which has the characteristics of long treatment time and large dosage of thrombolytic drugs. In the absence of good monitoring methods, problems such as low thrombolytic efficiency and high risk of bleeding are easy to occur. OBJECTIVE: To evaluate the value of D-dimer (D-D) and fibrinogen (FIB) testing as a thrombolysis-monitoring method during CDT for acute DVT. METHODS: Twenty patients with acute DVT were divided into group A and group B. During CDT, the D-D and FIB testing every 8 h were used in group A, and the venography and FIB testing every 24 h in group B. The thrombolysis rate, thrombolysis time, urokinase dosage, and X-ray radiation dose were compared. RESULTS: The thrombolysis rate in group A was significantly higher than that in group B (p < 0.05), but the number of venography and radiation dose were significantly lower than those in group B (p < 0.05). CONCLUSION: D-D and FIB testing can improve the thrombolysis rate, reduce the risk of bleeding, and decrease the number of angiograms and X-ray radiation dose during CDT.

Evidence that sensitization to triple-knockout pig cells will not be detrimental to subsequent allotransplantation.

The current evidence is that sensitization to a pig xenograft does not result in the development of antibodies that cross-react with alloantigens, and therefore, sensitization to a pig xenograft would not be detrimental to the outcome of a subsequent allograft. This evidence relates almost entirely to the transplantation of cells or organs from wild-type or α1,3-galactosyltransferase gene-knockout (GTKO) pigs. However, it is not known whether recipients of triple-knockout (TKO) pig grafts who become sensitized to TKO pig antigens develop antibodies that cross-react with alloantigens and thus be detrimental to a subsequent organ allotransplant. We identified a single baboon (B1317) in which no (or minimal) serum anti-TKO pig antibodies could be measured-in our experience unique among baboons. We sensitized it by repeated subcutaneous injections of TKO pig peripheral blood mononuclear cells (PBMCs) in the absence of any immunosuppressive therapy. After TKO pig PBMC injection, there was a transient increase in anti-TKO pig IgM, followed by a sustained increase in IgG binding to TKO cells. In contrast, there was no serum IgM or IgG binding to PBMCs from any of a panel of baboon PBMCs (n = 8). We conclude that sensitization to TKO pig PBMCs in the baboon did not result in the development of antibodies that also bound to baboon cells, suggesting that there would be no detrimental effect of sensitization on a subsequent organ allotransplant.

Co-modification of calcium phosphate cement to achieve rapid bone regeneration in osteoporotic femoral condyle defect with lithium and aspirin.

Local application of lithium or aspirin with biological scaffold has been identified as a potent means to improve bone formation. In this study, lithium and aspirin modified calcium phosphate cement (Asp-Li/CPC) was prepared, and the feasibility of this biological scaffold in the treatment of osteoporotic bone defect was observed in vivo and in vitro. In vitro experiments confirmed that Asp-Li/CPC had better ability to promote MC3T3-E1 cells differentiation into osteoblasts, osteoblast mineralization and viability, and promote cell expression of ALP, OP, RUNX-2, OC and COL-1 protein than simple CPC or lithium modified CPC by MTT, Alizarin red staining and Western blot evaluation. In vivo experiments confirmed that Asp-Li/CPC presented the strongest effect on bone regeneration and bone mineralization through the comparison with CPC group and Li/CPC group with X-ray images, Micro-CT and Histological evaluation. RT-qPCR analysis showed that Asp-Li/CPC, Li/CPC group and CPC group demonstrated increased BMP2, Smad1, OPG than the OVX group (P<0.05), while Asp-Li/CPC exhibited decreased TNF-α, IFN-γ and RANKL than the OVX group (P<0.05). Experiments in vivo and in vitro show that Asp-Li/CPC is a scheme for rapid repair of femoral condylar defects, and these effects may be achieved by inhibiting local inflammation and through BMP-2/Smad1 and OPG/RANKL signaling pathway.

Pig kidney xenotransplantation: Progress toward clinical trials.

Pig organ xenotransplantation offers a solution to the shortage of deceased human organs for transplantation. The pathobiological response to a pig xenograft is complex, involving antibody, complement, coagulation, inflammatory, and cellular responses. To overcome these barriers, genetic manipulation of the organ-source pigs has largely been directed to two major aims-(a) deletion of expression of the known carbohydrate xenoantigens against which humans have natural (preformed) antibodies, and (b) transgenic expression of human protective proteins, for example, complement- and coagulation-regulatory proteins. Conventional (FDA-approved) immunosuppressive therapy is unsuccessful in preventing an adaptive immune response to pig cells, but blockade of the CD40:CD154 costimulation pathway is successful. Survival of genetically engineered pig kidneys in immunosuppressed nonhuman primates can now be measured in months. Non-immunological aspects, for example, pig renal function, a hypovolemia syndrome, and rapid growth of the pig kidney after transplantation, are briefly discussed. We suggest that patients on the wait-list for a deceased human kidney graft who are unlikely to receive one due to long waiting times are those for whom kidney xenotransplantation might first be considered. The potential risk of infection, public attitudes to xenotransplantation, and ethical, regulatory, and financial aspects are briefly addressed.

Human anti-α-fucose antibodies are xenoreactive toward GGTA1/CMAH knockout pigs.

Progress has been made in overcoming antibody-mediated rejection of porcine xenografts by deleting pig genes that produce unique carbohydrate epitopes. Pigs deficient in galactose α-1,3 galactose (gene modified: GGTA1) and neu5Gc (gene modified: CMAH) have reduced levels of human antibody binding. Previously we identified α-fucose as a glycan that was expressed in high levels on cells of GGTA1/CMAH KO pigs. To validate the α-fucose phenotype observed previously we compared lectin affinity toward human and pig serum glycoproteins by dot blot analysis and confocal microscopy. Human anti-fucose antibody isolated by affinity chromatography was tested for specificity to L-fucose by custom macroarray. The affinity and cytotoxicity of the isolated human anti-fucose antibody toward human and GGTA1/CMAH KO pig PBMCs was determined by flow cytometry. Dot blot and confocal analysis support out previous findings that α-fucose is more highly expressed in pigs than humans. Pig kidney glomeruli and tubules contain abundant α-fucose and may represent focal sites for anti-α-fucose antibody binding. The Isolated human anti-fucose IgA, IgG and IgM bound to GGTA1/CMAH KO pig PBMC and were cytotoxic. Interestingly, the isolated human IgG cross reacted with the methyl pentose, L-rhamnose. Human anti-fucose antibody bound and was cytotoxic to GGTA1/CMAH KO pig peripheral blood monocytes. We have shown that α-fucose is an abundant target for cytotoxic human antibody in the organs of genetically modified pigs important to xenotransplantation.

The final obstacle to successful pre-clinical xenotransplantation?

Genetically engineered pigs are now available for xenotransplantation in which all three known carbohydrate xenoantigens, against which humans have natural antibodies, have been deleted (triple-knockout [TKO] pigs). Furthermore, multiple human transgenes have been expressed in the TKO pigs, all of which are aimed at protecting the cells from the human immune response. Many human sera demonstrate no or minimal antibody binding to, and little or no cytotoxicity of, cells from these pigs, and this is associated with a relatively low T-cell proliferative response. Unfortunately, baboons and other Old World NHPs have antibodies against TKO pig cells, apparently directed to a fourth xenoantigen that appears to be exposed after TKO. In our experience, most, if not all, humans do not have natural antibodies against this fourth xenoantigen. This discrepancy between NHPs and humans is providing a hurdle to successful translation of pig organ transplantation into the clinic, and making it difficult to provide pre-clinical data that support initiation of a clinical trial. The potential methods by which this obstacle might be overcome are discussed. We conclude that, whatever currently available genetically engineered pig is selected for the final pre-clinical studies, this may not be the optimal pig for clinical trials.

Role of serum cystatin C in the prediction of contrast-induced nephropathy after intra-arterial interventions.

BACKGROUND: The diagnosis of contrast-induced nephropathy (CIN) is usually based on changes in serum creatinine (sCr). However, sCr has poor sensitivity as a biomarker of kidney injury. The aim of this study was to investigate the usefulness of serum cystatin C (sCysC) to predict CIN after intra-arterial interventions. METHODS: A total of 360 consecutive patients underwent intra-arterial procedures using digital subtraction angiography. SCr, sCysC, and estimated glomerular filtration rate were measured at 1 to 2 days before and at 48, 72 h, and 7 days after the procedure. RESULTS: Thirty-one patients (8.61%) developed CIN. Receiver operating characteristic (ROC) curve analysis showed that pre-operative sCysC levels had good discriminatory power (area under the curve [AUC] = 0.634; 95% confidence interval [CI] = 0.526-0.743) for evaluating the risk of CIN after an endovascular procedure, with a sensitivity of 53.33% and specificity of 73.70%. ROC analysis showed that sCysC at 48 h after contrast medium administration was predictive of CIN after an endovascular procedure (AUC = 0.735; 95% CI = 0.647-0.822) with satisfactory sensitivity of 74.20% and specificity of 63.90%. Diabetes mellitus was an independent risk factor for CIN (odds ratio = 2.778; 95% CI = 1.045-7.382; P = 0.040). CONCLUSIONS: SCysC is an appropriate biomarker to predict the occurrence of CIN. Baseline sCysC before an intervention is useful to obtain a preliminary estimate of the risk of CIN. A 48-h cut-off value of sCysC of 0.99 mg/L after an endovascular procedure may help to rule out patients at lower risk of CIN.

Circ_0080425 inhibits cell proliferation and fibrosis in diabetic nephropathy via sponging miR-24-3p and targeting fibroblast growth factor 11.

Diabetic nephropathy (DN) is a severe end-stage kidney disease developed from diabetes mellitus. The involvement of circular RNAs (circRNAs) in modulating DN pathogenesis has been implied, but underlying mechanism is still lacking. In this study, we demonstrated that the expression of circ_0080425 correlated with the DN progression, and exerted positive effect on cell proliferation and fibrosis in mesangial cells. Further assessment suggested that circ_0080425 function as sponge harboring miR-24-3p. Moreover, miR-24-3p negatively correlated with the DN progression, and showed an antagonistic effect to circ_0080425on regulating MCs cell proliferation and fibrosis. Bioinformatics analysis predicted fibroblast growth factor 11 (FGF11) acting as direct downstream target of miR-24-3p. Indeed, the expression of FGF11 was significantly activated by circ_0080425 while suppressed by miR-24-3p. Knockdown of FGF11 resulted in a significant reduced cell proliferation rate and fibrosis. In addition, miR-24-3p inhibitor rescued the suppression of si-circ_0080425 on FGF11, suggesting that circ_0080425 competitive binding to miR-24-3p could release FGF11 from miR-24-3p suppression, which subsequently promoted DN progression.In conclusion, we have reported a novel circ_0080425-miR-24-3p-FGF11 axis, and explored the underlying mechanism in regulating DN pathogenesis.

LINC00205 modulates the expression of EPHX1 through the inhibition of miR-184 in hepatocellular carcinoma as a ceRNA.

Several studies have shown that low expression of epoxide hydrolase 1 (EPHX1) is closely associated with varying human cancers, including hepatocellular carcinoma (HCC). This study aims to explore the potential mechanism of EPHX1 silencing and revealed a novel regulatory pathway in the pathogenesis of HCC. In this study, micro ribonucleic acid (miR)-184 was predicted and validated to be a regulator of EPHX1 through experiments, and its expression was negatively correlated with the messenger RNA (mRNA) levels of EPHX1 in primary tumors. Elevation of EPHX1 suppressed cell proliferation and migration as well as cell cycle progression, and induced apoptosis, while downregulation of miR-184 exhibited the opposite effect on cellular processes. Moreover, LINC00205 interacted with miR-184 and was markedly downregulated in tumors. The effects of the miR-184 inhibitor on cell proliferation, apoptosis, and migration were reversed in part by the transfection with LINC00205 small interfering RNAs. In addition, LINC00205 acted as a molecular sponge to positively regulate the mRNA and protein levels of EPHX1 via regulating miR-184. The tumorigenicity of HCC cells was enhanced by LINC00205 shRNA but diminished by overexpression of EPHX1 in vivo. Clinically, the EPHX1 expression in patients with HCC was markedly downregulated. Taken together, the results of this study suggest that as a competing endogenous RNA, LINC00205 may regulate EPHX1 by inhibiting miR-184 in the progression of HCC and that targeting the LINC00205/miR-184/EPHX1 axis may provide a treatment protocol for patients.

HLA Class I-sensitized Renal Transplant Patients Have Antibody Binding to SLA Class I Epitopes.

BACKGROUND: Highly sensitized patients are difficult to match with suitable renal allograft donors and may benefit from xenotransplant trials. We evaluate antibody binding from sensitized patients to pig cells and engineered single allele cells to identify anti-human leukocyte antigen (HLA) antibody cross-species reactivity with swine leukocyte antigen (SLA). These novel testing strategies assess HLA/SLA epitopes and antibody-binding patterns and introduce genetic engineering of SLA epitopes. METHODS: Sensitized patient sera were grouped by calculated panel reactive antibody and luminex single antigen reactivity profile and were tested with cloned GGTA1/CMAH/B4GalNT2 glycan knockout porcine cells. Pig reactivity was assessed by direct flow cytometric crossmatch and studied following elution from pig cells. To study the antigenicity of individual class I HLA and SLA alleles in cells, irrelevant sera binding to lymphoblastoid cells were minimized by CRISPR/Cas9 elimination of endogenous class I and class II HLA, B-cell receptor, and Fc receptor genes. Native HLA, SLA, and mutants of these proteins after mutating 144K to Q were assessed for antibody binding. RESULTS: Those with predominately anti-HLA-B&C antibodies, including Bw6 and Bw4 sensitization, frequently have low pig reactivity. Conversely, antibodies eluted from porcine cells are more commonly anti-HLA-A. Single HLA/SLA expressing engineered cells shows variable antigenicity and mutation of 144K to Q reduces antibody binding for some sensitized patients. CONCLUSIONS: Anti-HLA antibodies cross-react with SLA class I in predictable patterns, which can be identified with histocompatibility strategies, and SLA class I is a possible target of genetic engineering.